The HIV-1 matrix protein forms an icosahedral shell associated with the inner membrane of the mature virus. The protein performs a number of important functions throughout the viral life cycle, including anchoring the transmembrane envelope protein on the surface of the virus and assisting in viral penetration. We have recently determined the structure of the HIV-1 matrix protein at high resolution using multidimensional NMR spectroscopy. Proposed research will address important aspects of matrix structure and function beyond those revealed by the matrix protein solution structure. l) The structure of the HIV-l matrix protein will be determined in two different crystalline lattices. We anticipate that these crystal structures will resolve differences between the two published NMR structures and also allow us to reconstruct different aspects of the intermolecular packing interactions within the matrix protein shell of the HIV- l virus. Monoclinic crystals of the HIV- 1 matrix protein have been grown and data collected to a resolution of 2.5 Angstroms. A trigonal crystal form has also been observed recently. Growth of this crystal form will be optimized and the structure solved. 2) The structure of the HTLV-II matrix protein in solution will be determined. This structure will be important in its own right, and will also allow us to compare the structures of highly diverged matrix protein from the lentiviral (HIV-l) and oncoviral (HTLV-II) classes of human retroviruses. This should provide a framework for understanding the folding and organization of matrix proteins in all retroviruses. Preliminary NMR studies with 15N isotopically labeled HTLV-II matrix protein have revealed that the protein will be amenable to high resolution structure determination. 3) Finally, two important functions of the HIV-l matrix protein will be addressed in biochemical experiments. a) Interactions between the HIV-1 matrix protein and the intracellular domain of the envelope protein will be examined to determine how the matrix protein recruits and stabilizes the envelope protein on the surface of the virus. b) We will test the hypothesis that C-terminal matrix sequences involved in viral penetration adopt different structures in the proteolyzed matrix protein (found in the mature, "penetration-competent" form of the virus) and the gag polyprotein (found in the immature, budding virus). Structural changes accompanying proteolytic processing of the matrix domain of gag will be monitored using CD spectroscopy. Subsequent NMR studies on minimal structured fusion proteins will allow detailed comparison of the matrix protein domain before and after proteolysis.